The publications and other materials used herein to illuminate the background of the invention, and in particular, cases to provide additional details respecting the practice, are incorporated by reference.
In vitro analytical methods that allow the detection of specific analyte molecules—small molecules, electrolytes, proteins, lipids, carbohydrates or nucleic acids—or even microorganisms such as bacteria, viruses, fungi or protozoan organisms—are essential for modern human and veterinary medicine, control of the environment, monitoring of food safety and all other fields of biological research. In many analytical techniques, detection is at least partly based on nucleic acid amplification. Examples of nucleic acid amplification techniques include but are not limited to the polymerase chain reaction (PCR) (Saiki et al., 1985), nucleic acid sequence based amplification (NASBA) (Compton, 1991), reverse transcription PCR, and real-time PCR (Higuchi et al., 1992 and 1993). While nucleic acid amplification techniques are used to detect nucleic acid analytes, techniques exist that combine a nucleic acid assay and a different ligand binding assay. In such combination methods, nucleic acid amplification is used in the detection of a molecule such as a protein or other non-nucleic acid molecule. Examples of combination methods that comprise a nucleic acid amplification step include the immuno-polymerase chain reaction (Niemeyer et al., 2005) and the proximity ligation assay (Fredriksson et al., 2002).
Analytical assays in general can be heterogeneous, which means that at least one of the assay components is bound to a solid support and at least one of the assay components is in solution, or homogeneous, which means that all assay components are in solution. Such analytical techniques often require several pipetting steps to combine a set of detection reagents with the samples. These pipetting steps, if performed manually, constitute an important source of analytical error, especially in the case of assays in which small variations in assay component concentrations cause significant error (such as competitive immunoassays), and require a considerable amount of hands-on time. If the pipetting steps are automated, as is often done, the errors due to human actions as well as the amount of manual work can be minimized but, on the other hand, expensive equipment is needed for liquid handling. To make analytical techniques, that have a step where a sample is combined with a set of detection reagents, simpler, faster, more reliable and applicable even outside a specialized laboratory environment, it is possible to pre-dispense the detection reagents into a container, after which excess water is removed and the reagents are stored in a dry form. The dry reagent container will subsequently act as a reaction vessel also. Thus, carrying out the assay requires a minimum of liquid handling steps: only a sample in a suitable volume of a suitable liquid needs to be added into a vessel that already contains the dried detection reagents. Upon sample addition, the dried reagents are again dissolved and the analysis can begin. Such all-in-one dried assay reagents have been applied to for example immunoassays (Lövgren et al., 1996) and PCR assays (Nurmi et al., 2001).
Many biological and chemical reagents are stable in a dry form. However, a problem that is sometimes encountered is that the composition of detection reagents, when dried as a mixture, is unstable even if all components, when dried individually, would be stable. This instability may, for example, be caused by one or more of the reagents acting upon one or more of the other reagents during drying or storage in a manner that adversely affects the stability of the reagents One way to overcome this problem is to place an insulating layer between different reagents and thereby prevent any adverse reactions between different assay components. This kind of an approach has been described in the prior art (WO9738311, Lövgren et al. 1996). By separating at least one of the reagents that take part in the unwanted reactions, the reagent mixture can be stabilized. However, this approach requires the addition of an insulating layer, which introduces some problems to the manufacturing process. First of all, the reagent added after the insulating layer has to be dispensed in as small a volume as possible in order to avoid dissolving the insulating layer. Small volume dispensing is quite prone to variation, so assay analytical error can increase as a result. Furthermore, in some applications, it may be impossible to find a suitable composition for an insulating layer, since some of the components of the insulating layer itself may adversely affect the detection assay. Thus the production of dry reagents would be simpler, less prone to error and more often possible, if no insulating layer was needed. Another problem encountered when drying assay reagents is the method of drying itself: usually, lyophilization is used. Lyophilization, however, is a technically more demanding process than air-drying and it would be advantageous to have a way of stabilizing assay reagents so that air-drying could be used to remove excess water from the reaction mixtures instead of lyophilization.
In some assays that comprise a nucleic acid amplification step utilizing a nucleic acid polymerase, it is possible to dry the reagents as a mixture without any unwanted side reactions. An example of a fully functional assay in which all reagents are dried as a mixture has been published by for example Nurmi et al. (2001). However, in other cases, some nucleic acid polymerases exhibit unwanted side reactions or are otherwise unstable when they are dried together with other assay reagents. To solve this problem, a method is needed that allows stabilization of such reagent mixtures.